Penelitian ini bertujuan untuk mengetahui pengaruh substitusi daging sapi dengan daging ikan tuna terhadap kadar kolesterol dan asam lemak omega-3 bakso. Sebanyak 1.000 g adonan bakso dibuat dengan komposisi 600 g daging sapi, 200 g tepung tapioka, 160 g es batu, 20 g garam halus, 12 g bawang putih, 5 g lada halus, dan 3 g putih telur. Daging sapi disubstitusi dengan daging ikan tuna, dengan perlakuan 0%, 15%, dan 30%. Seluruh bahan digiling halus membentuk adonan, kemudian dibentuk pentolan (± 2 cm), direbus dalam air mendidih sampai matang, Parameters yang diuji, yaitu kadar kolesterol dan kandungan asam lemak omega-3 bakso. Data yang diperoleh dianalisis ragam menggunakan rancangan acak lengkap. Perbedaan antar perlakuan diuji lanjut menggunakan uji Duncan. Hasil penelitian menunjukkan bahwa substitusi daging sapi dengan daging ikan tuna menurunkan (P<0,01) kadar kolesterol dan meningkatkan (P<0,01) total asam lemak omega-3 bakso. Bakso dengan kadar kolesterol terendah, tetapi mempunyai total asam lema omega-3 tertinggi ada pada bakso yang disubstitusi dengan 30% daging ikan tuna. Dapat disimpulkan bahwa makin tinggi level daging ikan tuna yang digunakan untuk mensubstitusi daging sapi, dapat menurunkan kadar kolesterol, tetapi meningkatkan kadar total asam lemak omega-3 bakso daging sapi. Level terbaik daging ikan tuna untuk mensubstitusi daging sapi adalah 30%. ABSTRACT This study aims to determine the effect of substitution of beef with tuna meat on cholesterol and omega-3 fatty acid of meatballs. A total of 1,000 g of meatball dough is made with a composition of 600 g beef, 200 g tapioca flour, 160 g ice cubes, 20 g fine salt, 12 g garlic, 5 g fine pepper, and 3 g egg whites. Beef was substituted with tuna meat, with treatments 0%, 15%, and 30%. All ingredients are finely ground to form a dough, then formed a meatball (± 2 cm), boiled in boiling water until cooked. The parameters tested, namely cholesterol levels and omega-3 fatty acid content of meatballs. The data obtained were analyzed using a complete random design. The differences between treatments were further tested using the Duncan test. The results showed that the substitution of beef with 40% tuna meat and tapioca flour with purslane flour with different levels, decreasing (P<0.01) cholesterol levels and increasing (P<0.01) total omega-3 fatty acids of meatballs. The meatballs with the lowest cholesterol, but the highest total omega-3 are meatballs with substituted by 30% tuna meat. It can be concluded that the higher the level of tuna meat used to substitute beef, can decrease cholesterol content, but increase the total level of omega-3 fatty acids in beef meatballs. The best level of tuna meat to substitute beef is 30%.